*Purpose: Conventional therapy for liver transplant rejection (corticosteroids and ATG) has remained unchanged for six decades, and is not infrequently met with treatment failure. Here, we used single cell RNAseq with V(D)J profiling to provide novel insights into the mechanisms of liver transplant rejection and treatment failure, with a focus on defining expanded (ie, alloreactive) T cells and their gene expression profiles.

*Methods: Single cell suspensions were created from cryopreserved 16G liver biopsy tissue using cold active proteases. Whole cell and TCR libraries were constructed using 10X Chromium 5’v1.1 and 5’V(D)J TCR sequencing kits. Cells were clustered based on cell type specific gene expression profiles by unsupervised analysis in Seurat and TCR libraries were superimposed into the cell clusters. TCR clonotypes were defined as expanded if the TCR sequence was expressed in >2 cells and in >0.5% of the total barcodes.

*Results: 3,777 cells isolated from 8 biopsies obtained from 4 pediatric patients, who were >1 year post-transplant at time of rejection were analyzed (Table 1). In integrated analysis of one patient with steroid-resistant rejection and two patients with steroid-responsive ACR, 618 TCR clonotypes were identified, 14 (2.26%) of which were clonally expanded. Analysis of 3 serial biopsies from a fourth patient yielded 524 TCR clonotypes of which 26 (4.96%) were expanded. All clonally expanded cells were genetically identified as CD8⁺ tissue resident memory Tcells (Trm; CD69, Eomes, KLRG1, IFN). The majority of expanded clones were resistant to ATG depletion. In contrast, CD4⁺ T cells were only found in the unexpanded T cell pool and had a gene expression profile consistent with regulatory T cells (Foxp3, CTLA4, CD70).

*Conclusions: Despite 6-antigen HLA mismatches corresponding to a massive number of potential alloantigens, we uncovered only 40 clonally-expanded CD8⁺ T cells. These clones persisted over time, suggesting that the limited clonal expansion is not due to sampling bias, but rather to a highly focused alloresponse. Transcriptomic analysis revealed that expanded clones were characterized as Trm cells while unexpanded clones possessed a regulatory phenotype.

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